Background: Induced pluripotent stem cells (iPSCs) are human somatic cells that have been reprogrammed to a pluripotent state. Several methods have been used to generate hepatocyte-like cells from iPSCs. However, these hepatic cells have limited clinical application because of their immature function compared to primary hepatocytes. Mesenchymal stem cells (MSCs) have been reported to inhibit apoptosis of hepatic cells and to improve hepatic regeneration in acute liver injury. Therefore, we expected that MSCs had the potential to positively contribute to the maturation of hepatic cells. Here we demonstrate the effect of MSCs on the maturation of hepatoblasts derived from human iPSCs. Methods: MSCs were isolated from human bone marrow and cultured to 70-80% confluence. MSC-conditioned medium (MSC-CM) was collected 48 h after culture in hepatic maturation medium. Human iPSC-derived hepatoblasts were then cultured for 6 days with MSC-CM. Hepatic functions were analyzed and compared to those from cells cultured in general maturation medium. Results: Cells in both groups had a cuboidal morphology typical of hepatocytes. The proportion of Oct4-positive cells was decreased and those of albumin- and alpha-fetoprotein-positive cells were increased in the MSC-CM group. Albumin secretion and urea synthesis as well as cytochrome P450 (CYP) 3A4 activity were enhanced in the MSC-CM group. The gene expressions of some CYP enzymes were upregulated as demonstrated by RT-PCR. Conclusion: Secreted molecules from human MSCs could enhance the hepatic function of human iPSC-derived hepatocyte-like cells. Although more technological innovations are needed, MSC-CM will be useful as a novel efficient strategy for clinically relevant hepatic cell maturation.